Enzymatic Preparation of D.P.N. H2 and Determination D.P.N.

Bonnichsen, Roger; Carlsson, Christine; Webb, M.E.; Rottenberg, Max

doi:10.3891/acta.chem.scand.04-0714

Cited by 32 publications

(6 citation statements)

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“…For use enzyme samples were centrifuged, the enzyme was dissolved and dialysed against three changes of phosphate buffer (ionic strength 0.1, pH 7.4) for 3 days at 4 "C. The enzyme was centrifuged and the activesite concentration (pN) assayed as described previously [14]. The apparent purity was 87--93O/, when an absorption coefficient ( A : T$ml) a t 280 nm of 0.45 [15] was used to determine protein concentration (pM).Nucleotides and pyridoxal-P were purchased from Sigma Chemical Co. (St. Louis, Mo.). Imidazole was recrystallized as described previously [ 141.…”

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confidence: 99%

“…Plapp [3] has shown that methyl picolinimidate reacts with 50 of the 60 lysine residues in the enzyme, resulting in a large activation of enzyme activity when assayed a t pH 9.0 with high concent<rations of NAD+ and ethanol. I n this study 4-Biphenyl-carboxylic acid was purchased from Fluka AG (Buchs, Switzerland) and recrystallised from methanol and then methanol-water ; melting point 223 -225 "C. Methyl picolinimidate was synthesised as described previously [15]. Other chemicals were of reagent grade.…”

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The Lysines in Liver Alcohol Dehydrogenase

McKinley-McKee¹,

Morris²

1972

European Journal of Biochemistry

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Liver alcohol dehydrogenase is reversibly inactivated by pyridoxal 5'-phosphate. When the enzyme is saturated with pyridoxal-P the residual activity is about 2001,. The enzyme can be completely inactivated by successive covalent labelling with pyridoxal-P, using sodium borohydride reduction. A maximum of about 11 pyridoxal-P groups per subunit were covalently incorporated into the enzyme.The binding to the enzyme of several known inhibitors was investigated by studying their effect on the reversible inactivation of the enzyme by pyridoxal-P a t pH 8.0. The effect of these ligands on the activation of the enzyme by methyl picolinimidate was also studied. AMP protected competitively against inactivation by pyridoxal-P and also protected strongly against activation by methyl picolinimidate. After picolinimidylation of the enzyme the binding of AMP a t pH 10 was 8-fold weaker.AMP and NADH both protected one lysine residue per subunit from reaction with pyridoxal-P . It is concluded that there is one lysine residue a t the active centre of the enzyme and that this lysine plays an important part in binding the coenzyme by attracting the phosphate group of the AMP moiety of the coenzyme. Coenzyme binding to the picolinimidylated enzyme is weakened by steric interference between the picolinomidylated active-centre lysine and the AMP moiety of the coenzyme. Since effects of neutral zinc-binding ligands cannot be explained by steric or electrostatic effects, it is suggested that they produce conformational changes in the enzyme, perhaps by binding a t a site other than the active-centre zinc atom.

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confidence: 99%

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confidence: 99%

The Lysines in Liver Alcohol Dehydrogenase

McKinley-McKee¹,

Morris²

1972

European Journal of Biochemistry

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“…Reduced TPN was prepared chemically by reduction of the oxidized nucleotide with dithionite (KAPLAN et al 1952). Reduced DPN was prepared by reduction of oxidized DPN with the alcohol-alcoholdehydrogenase system (BONNICHSEN 1950).…”

Section: Methodsmentioning

confidence: 99%

On the Metabolism of Lidocaine I. The Properties of the Enzyme System responsible for the oxidative Metabolism of Lidocaine.

Hollunger¹

1961

Acta Pharmacologica et Toxicologica

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“…Alcohol dehydrogenases from yeast or from horse liver (30,210,278) are particularly convenient. At pH 10 the reduction of DPN by these enzymes is complete even in the absence of carbonyl fixatives, provided that excess substrate is present (29). DPNH is readily determined by the same enzymes in the presence of excess acetaldehyde, but in the measurement of DPNH oxidation the pH is kept around neutrality. "…”

Section: Estimationmentioning

confidence: 99%

“…DPN or COIII tCysteinesulfinate + "eluate factor" + OZ + H20 -pyruvate + sulfate + aspart,ate (29) Reaction (23) depicts the action of a DPN-requiring amino acid dehydrogenase, which has been extracted from rat liver mitochondria1 acetone powder (269) and appears to be specific for L-cysteinesulfinic acid. 8-Sulfinylpyruvate, the product of reaction (21) is further metabolized to pyruvate by way of reactions (25) and (26).…”

Section: Sulfur Metabolismmentioning

confidence: 99%